Somatostatin agonists

ABSTRACT

The present invention is directed to cyclic peptides of formula (I): X-A 1 -cyclo(D-Cys-A 3 -A 4 -Lys-A 6 -A 7 )-A 8 -Y, or a pharmaceutically acceptable salt thereof. The peptides bind selectively to the somatostatin subtype receptor type-5 and elicit an agonist effect from the somatostatin subtype receptors. The peptides are useful for treating a variety of diseases, including Cushings Syndrome, gonadotropinoma, hyperparathyroidism, Paget&#39;s disease, VIPoma, nesidioblastosis, hyperinsulinism, gastrinoma, Zollinger-Ellison Syndrome, hypersecretory diarrhea related to AIDS and other conditions, irritable bowel syndrome, pancreatitis, Crohn&#39;s Disease, systemic sclerosis, thyroid cancer, psoriasis, hypotension, panic attacks, sclerodoma, small bowel obstruction, gastroesophageal reflux, duodenogastric reflux, Graves&#39; Disease, polycystic ovary disease, upper gastrointestinal bleeding, pancreatic pseudocysts, pancreatic ascites, leukemia, meningioma, cancer cachexia, acromegaly, restenosis, hepatoma, lung cancer, melanoma, inhibiting the accelerated growth of a solid tumor, decreasing body weight, treating insulin resistance, Syndrome X, prolonging the survival of pancreatic cells, fibrosis, hyperlipidemia, hyperamylinemia, hyperprolactinemia and prolactinemia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national filing under 35 U.S.C. §371 ofinternational (PCT) application No. PCT/US00/17401, filed Jun. 23, 2000,designating the U.S., and claiming priority to U.S. provisionalapplication Ser. No. 60/141,028, filed Jun. 25, 1999.

BACKGROUND OF THE INVENTION

The present invention is directed to cyclic peptides that havesomatostatin agonist activity, as defined by formula (I), shown anddefined hereinbelow, or a pharmaceutically acceptable salt thereof,pharmaceutical compositions comprising said peptides and the use thereofas a somatostatin receptor subtypes agonist. The peptides of the presentinvention bind selectively to the somatostatin subtype receptor 5 andelicit an agonist effect from the somatostatin subtype receptors thatthe peptides bind to.

Somatostatin (SRIF) is a cyclic tetradecapeptide hormone containing adisulfide bridge between position 3 and position 14 (Heiman, et al.,Neuroendocrinology, 45:429-436 (1987)) and has the properties ofinhibiting the release of growth hormone (GH) and thyroid-stimulatinghormone (TSH), inhibiting the release of amylin, insulin and glucagon,reducing gastric secretion and neurotransmitter release. Metabolism ofsomatostatin by aminopeptidases and carboxypeptidases leads to a shortduration of action. Because of the short half-life of the nativesomatostatin, various somatostatin analogs have been developed, e.g.,for the treatment of acromegaly. Raynor, et al., Molecular Pharmacol.43:838 (1993).

Five distinct somatostatin receptors have been identified andcharacterized. Hoyer, et al., Naunyn-Schmiedeberg's Arch. Pharmacol.,350:441 (1994). Somatostatin binds to five distinct receptor (SSTR)subtypes with relatively high and equal affinity for each subtype.Binding to the different types of somatostatin subtypes have beenassociated with the treatment of the following conditions and/ordiseases. (“SSTR-2”) (Raynor, et al., Molecular Pharmacol. 43:838(1993); Lloyd, et al., Am. J. Physiol. 26B:G102 (1995)) while theinhibition of insulin has been attributed to the somatostatin type-5receptor (“SSTR-5”) (Coy, et al. 197:366-371 (1993)). Activation oftypes 2 and 5 have been associated with growth hormone suppression andmore particularly GH secreting adenomas (Acromegaly) and TSH secretingadenomas. Activation of type 2 but not type 5 has been associated withtreating prolactin secreting adenomas. Other indications associated withactivation of the somatostatin subtypes are inhibition of insulin and/orglucagon and more particularly diabetes mellitus, angiopathy,proliferative retinopathy, dawn phenomenon and Nephropathy; inhibitionof gastric acid secretion and more particularly peptic ulcers,enterocutaneous and pancreaticocutaneous fistula, irritable bowelsyndrome, Dumping syndrome, watery diarrhea syndrome, AIDS relateddiarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitisand gastrointestinal hormone secreting tumors; treatment of cancer suchas hepatoma; inhibition of angiogenesis, treatment of inflammatorydisorders such as arthritis; retinopathy; chronic allograft rejection;angioplasty; preventing graft vessel and gastrointestinal bleeding. Itis preferred to have an analog which is selective for the specificsomatostatin receptor subtype responsible for the desired biologicalresponse, thus, reducing interaction with other receptor subtypes whichcould lead to undesirable side effects.

The peptides of formula (I) are a sub-genus encompassed by a genus ofcompounds described and claimed in U.S. application Ser. No. 08/855,204,filed May 13, 1997, now U.S. Pat. No. 6,262,229, issued Jul. 17, 2001and assigned in part to the assignee of the present invention. Thecompounds of formula (I) of the present application are not specificallydescribed in U.S. Pat. No. 6,262,229. It has been unexpectedly andsurprisingly discovered that the compounds of formula (I) of the presentinvention possess somatostatin agonist activity. This is an unexpectedand surprising discovery since the compounds of U.S. Pat. No. 6,262,229were originally found to possess somatostatin antagonist activity.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a peptide of theformula (I),X-A¹-cyclo(D-Cys-A³-A⁴-Lys-A⁶-A⁷)-A⁸-Y.  (I)or a pharmaceutically acceptable salt thereof,wherein

-   A¹ and A³ are each independently the D- or L-isomer of an amino acid    selected from the group consisting of Phe, Tyr, Tyr(I), Trp, 3-Pal,    4-Pal, Cpa and Nal;-   A⁴ is L-Trp, D-Trp, L-β-methyl-Trp or D-β-methyl-Trp;-   A⁶ is —NH—(CHR¹)_(n)—CO—, where n is 2, 3, or 4;-   A⁷ is L- or D-Cys;-   A⁸ is the D- or L-isomer of an amino acid selected from the group    consisting of Phe, Tyr, Tyr(I), Trp, Nal, Cpa, Val, Leu, Ile, Ser    and Thr;-   Y is NR²R³ where R² and R³ are each independently H or (C₁-C₅)alkyl;-   R¹ is selected from the group consisting H, (C₁-C₄)alkyl and    —CH₂-aryl; wherein said aryl is an optionally substituted moiety    selected from the group consisting of phenyl, 1-naphthyl, and    2-naphthyl, wherein said optionally substituted moiety is optionally    substituted with one or more substituents each independently    selected from the group consisting of (C₁₋₆)alkyl, (C₂₋₆)alkenyl,    (C₂₋₆)alkynyl, aryl, aryl(C₁₋₆)alkyl, (C₁₋₆)alkoxy, —N(R⁴R⁵), —COOH,    —CON(R⁴R⁵), halo, —OH, —CN, and —NO₂;-   R⁴ and R⁵ each is, independently for each occurrence, H or    (C₁₋₃)alkyl;-   where the Cys of A² is bonded to the Cys of A⁷ by a di-sulfide bond    formed from the thiol groups of each Cys.

A preferred group of peptides of the foregoing peptide of formula (I) iswherein

-   X is H;-   A¹ is L-Phe, D-Phe, L-Cpa or D-Cpa;-   A³ is L-Tyr, L-Trp or L-3-Pal;-   A⁴ is D-Trp;-   A⁶ is β-Ala or Gaba;-   A⁷ is L-Cys;-   A⁸ is Thr, L-Trp, L-Leu or L-Nal; and-   R² and R³ are each H; or a pharmaceutically acceptable salt thereof.

Preferred peptides of the immediately foregoing group of peptides are:

-   Cpa-cyclo(D-Cys-3-Pal-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;-   Cpa-cyclo(D-Cys-3-Pal-D-Trp-Lys-β-Ala-Cys)-Nal-NH₂;-   Phe-cyclo(D-Cys-3-Pal-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;-   Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;-   Phe-cyclo(D-Cys-Trp-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;-   Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Trp-NH₂;-   D-Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;-   D-Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Leu-NH₂; and-   Phe-cyclo-(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Thr-NH₂; or a    pharmaceutically acceptable salt thereof.

Preferred peptides of the immediately foregoing group of peptides are:

-   Cpa-cyclo(D-Cys-3-Pal-D-Trp-Lys-Gaba-Cys)-Nal-NH₂; and-   Cpa-cyclo(D-Cys-3-Pal-D-Trp-Lys-β-Ala-Cys)-Nal-NH₂; or a    pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a pharmaceuticalcomposition useful for eliciting a somatostatin agonist response in ahuman or other animal which comprises an effective amount of a peptideof formula (I) or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

In yet another aspect, the present invention provides a method ofeliciting a somatostatin agonist response in a human or other animal inneed thereof, which comprises administering an effective amount of apeptide of formula (I) or a pharmaceutically acceptable salt thereof tothe human or other animal.

In a further aspect, the present invention provides a method ofselectively binding a somatostatin subtype receptor type 5 in a human orother animal, which comprises administering an effective amount of apeptide of formula (I) or a pharmaceutically acceptable salt thereof tothe human or other animal.

In still a further aspect, the present invention provides a method oftreating a disease or condition in a human or other animal in needthereof, which comprises administering an effective amount of a peptideof formula (I) or a pharmaceutically acceptable salt thereof to thehuman or other animal, wherein said disease or condition is selectedfrom the group consisting of Cushings Syndrome, gonadotropinoma,hyperparathyroidism, Paget's disease, VIPoma, nesidioblastosis,hyperinsulinism, gastrinoma, Zollinger-Ellison Syndrome, hypersecretorydiarrhea related to AIDS and other conditions, irritable bowel syndrome,pancreatitis, Crohn's Disease, systemic sclerosis, thyroid cancer,psoriasis, hypotension, panic attacks, sclerodoma, small bowelobstruction, gastroesophageal reflux, duodenogastric reflux, Graves'Disease, polycystic ovary disease, upper gastrointestinal bleeding,pancreatic pseudocysts, pancreatic ascites, leukemia, meningioma, cancercachexia, acromegaly, restenosis, hepatoma, lung cancer, melanoma,inhibiting the accelerated growth of a solid tumor, decreasing bodyweight, treating insulin resistance, Syndrome X, prolonging the survivalof pancreatic cells, fibrosis, hyperlipidemia, hyperamylinemia,hyperprolactinemia and prolactinemia.

In still a further aspect, the present invention provides a method ofinhibiting the secretion of growth hormone, insulin, glucagon orpancreatic exocrine secretion in a human or other animal in needthereof, which comprises administering a peptide of formula (I) or apharmaceutically acceptable salt thereof to said human or other animal.

In an even further aspect, the present invention provides a method ofimaging cells containing somatostatin receptors in vivo in a human orother animal, which comprises administering a peptide of formula (I),provided that at least one of A¹, A³ or A⁸ is Tyr(I), or apharmaceutically acceptable salt thereof to said human or other animal.

In another aspect, the present invention provides a method of imagingcells containing somatostatin receptors in vitro, which comprisesadministering a peptide of formula (I), provided that at least one ofA¹, A³ or A⁸ is Tyr(I), or a pharmaceutically acceptable salt thereof tosaid human or other animal. Such peptides of the present invention canbe used either in vivo to detect cells having somatostatin receptors(e.g., cancer cells) or in vitro as a radioligand in a somatostatinreceptor binding assay.

The three letter abbreviations accepted in the art are used to refer tothe amino acids in a peptide of the present invention. In the formulaset forth herein, the disulfide bond between the thiol group on the sidechain of residue A₂ (i.e., D-Cys) and the thiol group on the side chainof residue A₇ (i.e., L-Cys or D-Cys) is not shown. The following aminoacid abbreviations stand for the name indicated next to it:Cpa=p-chlorophenylalanine; Nal=β-(2-naphthyl)alanine;3-Pal=β-(3-pyridyl)-alanine; 4-Pal=β-(4-pyridyl)-alanine; andGaba=4-aminobutyric acid. The definition of “—NH—(CH₂)_(n)—CO— where nis 2, 3, or 4” encompasses such amino acids as β-Ala and Gaba.

Unless noted otherwise, the three letter abbreviation of an amino acidrefers to the L-isomer.

The term alkyl is intended to include those alkyl groups of thedesignated length in either a straight or branched configuration.Exemplary of such alkyl groups are methyl, ethyl, propyl, isopropyl,butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, and the like. Whenthe term C₀-alkyl is included in a definition it is intended to denote asingle covalent bond.

The term alkenyl is intended to include hydrocarbon groups having one ormore double bonds and the designated number of carbon atoms in either astraight or branched configuration. Exemplary of such alkenyl groups areethenyl, propenyl, isopropenyl, butenyl, sec-butenyl, tertiary butenyl,pentenyl, isopentenyl, hexenyl, isohexenyl and the like.

The term alkynyl is intended to include those alkynyl groups, i.e.,hydrocarbon groups having one or more triple bonds, having thedesignated number of carbon atoms in either a straight or branchedconfiguration. Exemplary of such alkynyl groups are ethynyl, propynyl,butynyl, pentynyl, isopentynyl, hexynyl, isohexynyl and the like.

The term alkoxy is intended to include those alkoxy groups having thedesignated number of carbon atoms in either a straight or branchedconfiguration. Exemplary of such alkoxy groups are methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy,isopentoxy, hexoxy, isohexoxy and the like.

The term aryl is intended to include aromatic rings known in the art,which can be mono-cyclic or bi-cylic, such as phenyl and naphthyl.

The term halo is intended to include chlorine, bromine, iodine, andfluorine.

DETAILED DESCRIPTION

One skilled in the art can, based on the description herein, utilize thepresent invention to its fullest extent. The following specificembodiments are, therefore, to be construed as merely illustrations ofthe invention and is not meant to be construed as limiting the fullscope of the invention.

Peptides of the present invention can be and were synthesized on RinkAmide MBHA resin,(4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-MBHAresin), using a standard solid phase protocol for FMOC chemistry andcleaved from the resin with a TFA/Phenol/H₂O/triisopropylsilane (83 ml/5g/10 ml/2 ml) mixture. Peptides were cyclized in CH₃CN/H₂O (5 ml/5 ml)using EKATHIOX™ resin (EKAGEN Corporation, San Carlos, Calif.) andpurified on C₁₈ silica (Rainin Instruments Co., Woburn, Mass. now VarianAnalytical, Walnut Creek, Calif.), using acetonitrile/0.1%trifluoroacetic acid buffers. Homogeneity was assessed by analyticalHPLC and were determined to be >95% for each peptide. Peptides werecharacterized by mass spectrometry.

The synthesis of iodinated Tyr (Tyr(I)) peptides of formula (I) of thepresent invention (e.g., the chloramine-T method) is well documented andare within the ability of a person of ordinary skill in the art. See,e.g., Czernick, et al., J. Biol. Chem. 258:5525 (1993) and EuropeanPatent No. 389,180 B1.

A peptide of formula (I) wherein X is

or

can be synthesized according to the processes and teachings of U.S. Pat.No. 5,552,520, the contents of which are incorporated herein in itsentirety.

Below is a detailed description of the synthesis of Examples 1 and 2.Other peptides within a compound of formula (I) can be prepared bymaking appropriate modifications, well-known to one of ordinary skill inthe art of peptide synthesis.

EXAMPLE 1

Step 1=Preparation ofFmoc-Cpa-S-trityl-D-Cys-Pal-N-in-t-Boc-D-Trp-N-ε-t-Boc-Lys-β-Ala-S-trityl-Cys-Nal-4-(2′,4′-Dimethoxyphenylaminomethyl) phenoxy-acetamido-norleucyl-4-methylbenzhydrylamine resin.

Rink amide MBHA resin (Novabiochem, Inc., San Diego, Calif.) 0.5 g,(0.265 mmole), was placed in a reaction vessel of a 24-RV peptidesynthesizer, assembled by connecting a shaker (from the BurrellWrist-Action Laboratory Shaker), a solvent distributor and a vacuumpump. The peptide synthesizer was programmed to perform the followingreaction cycle:

-   -   a. Dimethylformamide;    -   b. 25% piperidine in dimethylformamide (manually added) (2 times        for 15 minutes each with 1 time wash with DMF in between);    -   c. DMF washes (3×10 mL, 1 minute each);        The resin was stirred with FMOC-Nal (1.06 mmol),        2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate (HBUT) 1.007 mmole), and diisopropylethyl        amine (2.12 mmole) in dimethylformamide for about 1½ hours and        the resulting amino acid resin was then cycled through steps (a)        to (c) in the above washing/deblocking program.

The following amino acids were coupled successively to the Nal-resin bythe same procedure: Fmoc-S-Trityl-Cys, Fmoc-β-Ala, N-ε-t-Boc-Lys,Fmoc-(N-in-t-Boc)-D-Trp, Fmoc-Pal, Fmoc-S-trityl-D-Cys, andFmoc-p-Cl-Phe.

After washing with DMF (3×10 mL, about 1 minute each) and drying undervacuum, the complete peptide resin weighed 0.749 g.

Step 2: Preparation ofH-Cpa-cyclo(D-Cys-Pal-D-Trp-Lys-β-Ala-Cys)-Nal-NH₂

The peptide resin obtained from Step 1 of Example 1 (0.36 g, 0.087mmole) was mixed with a freshly prepared solution of TFA (8.8 mL),phenol (0.5 g), H₂O (0.5 mL) and triisopropylsilane (0.2 mL) at roomtemperature and stirred for about 2½ hours. Excess TFA was evaporatedunder reduced pressure to yield an oily residue. Ether was then added tothe oily residue and the free linear peptide was precipitated, filtered,and washed with dry ether. The crude peptide was then dissolved in 10 mLof CH₃CN/H₂O (5 mL/5 mL), followed by the addition of 200 mg EKATHIOX™resin. The mixture was stirred overnight and filtered. The filtrate wasevaporated to a small volume then applied to a column (22-250 mm) ofmicrosorb octadecylsilane silica (5 μm). Elution with a linear gradient(20% to 40%, over 60 minutes) of acetonitrile in water, (both solventshave 0.1% trifluoroacetic acid) yields fractions which were examined byanalytical high performance liquid chromatography (“HPLC”) and pooled togive maximum purity. Lyophilization of the solution from water gave 26mg of the product as white, fluffy powder. The product was found to behomogeneous by HPLC C₁₈ silica using the same eluant as described aboveand a linear gradient (30% to 70%, over 15 min) (Retention Time—6.313minutes). Infusion mass spectrometry confirmed the composition of thecyclic octapeptide, MW 1133.8.

EXAMPLE 2

Step 1: Preparation ofFmoc-Cpa-S-trityl-D-Cys-Pal-in-t-Boc-D-Trp-N-ε-t-Boc-Lys-Gaba-S-trityl-Cys-Nal-4-(2′,4′-Dimethoxyphenylaminomethyl)Phenoxyacetamido-norleucyl-4-methylbenzhydrylamine resin

Rink amide MBHA resin (Novabiochem, Inc. San Diego, Calif.) 0.2 g,(0.106 mmole) was placed in reaction vessel #3, (RV-3) of the 24-RVpeptide synthesizer. The peptide synthesizer was programmed to performthe following reaction cycle:

-   -   a. Dimethylformamide;    -   b. 25% piperidine in dimethylformamide (manually added) (2 times        for 15 minutes each with 1 time wash with DMF in between);    -   c. DMF washes (3×10 mL, 1 minute each);        The resin was stirred with FMOC-Nal (0.424 mmol),        2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate (HBUT) 0.403 mmole), and diisopropylethyl        amino (0.848 mmole) in dimethylformamide for about 1½ hours and        the resulting amino acid resin was then cycled through steps (a)        to (c) in the above wash program.

The following amino acids were coupled successively to the peptide resinby the same procedure: Fmoc-S-Trityl-Cys, Fmoc-Gaba, N-ε-t-Boc-Lys,Fmoc-N-in-t-Boc)-D-Trp, Fmoc-Pal, Fmoc-S-trityl-D-Cys, and Fmoc-Cpa.After washing with DMF (3×10 mL, about 1 minute each) and drying undervacuum, the complete resin weighed 0.31 g.

Step 2: Preparation of H-Cpa-cyclo(D-Cys-Pal-D-Trp-Lys-Gaba-Cys)-Nal-NH₂

The peptide resin obtained from Step 1 of Example 2 was mixed with afreshly prepared solution of TFA (8.3 mL), phenol (0.5 g), H₂O (1 mL)and triisopropylsilane (0.2 mL) at room temperature and stirred forabout 2½ hours. Excess TFA was evaporated under reduced pressure to givean oily residue. Ether was then added to the oily residue and the freelinear peptide was precipitated, filtered, and then washed with dryether. The crude peptide was then dissolved in 10 mL of CH₃CN/H₂Ofollowed by the addition of 200 mg of EKATHIOX™ resin. The mixture wasstirred overnight and filtered. The filtrate was evaporated to a smallvolume then applied to a column (22-250 mm) of microsorb octadecylsilanesilica (5 μm), and eluted with a linear gradient (20% to 100%, over 60minutes) of acetonitrile in water, in which both solvents have 0.1%trifluoroacetic acid. Fractions were examined by analytical highperformance liquid chromatography (“HPLC”) and pooled to give maximumpurity. Lyophilization of the solutions from water gave 13 mg of theproduct as white, fluffy powder. The product was found to be homogeneousby HPLC C₁₈ silica using the same eluant as described above (20% to 80%,over 15 min) (Retention time—9.195 minutes). Infusion mass spectrometryconfirmed the composition of the cyclic octapeptide, MW 1147.83.

The peptides of this invention can be provided in the form ofpharmaceutically acceptable salts. Examples of such salts include, butare not limited to, those formed with organic acids (e.g., acetic,lactic, maleic, citric, malic, ascorbic, succinic, benzoic,methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids(e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), andpolymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic,polyglycolic, or copolymers of polylactic-glycolic acids). A typicalmethod of making a salt of a peptide of the present invention is wellknown in the art and can be accomplished by standard methods of saltexchange. Accordingly, the TFA salt of a peptide of the presentinvention (the TFA salt results from the purification of the peptide byusing preparative HPLC, eluting with TFA containing buffer solutions)can be converted into another salt, such as an acetate salt bydissolving the peptide in a small amount of 0.25 N acetic acid aqueoussolution. The resulting solution is applied to a semi-prep HPLC column(Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1N ammoniumacetate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueoussolution for 0.5 hrs. and (3) a linear gradient (20% to 100% of solutionB over 30 min.) at a flow rate of 4 ml/min (solution A is 0.25N aceticacid aqueous solution; solution B is 0.25N acetic acid inacetonitrile/water, 80:20). The fractions containing the peptide arecollected and lyophilized to dryness.

The affinity of a peptide of the present invention for humansomatostatin subtype receptors 1 to 5 (sst₁, sst₂, sst₃, sst₄ and sst₅,respectively) is determined by measuring the inhibition of(¹²⁵I-Tyr¹¹)SRIF-14 binding to CHO-K1 transfected cells.

The human sst₁ receptor gene was cloned as a genomic fragment. A 1.5 KbPstI-XmnI segment containing 100 bp of the 5′-untranslated region, 1.17Kb of the entire coding region, and 230 bp of the 3′-untranslated regionwas modified by the Bg1II linker addition. The resulting DNA fragmentwas subcloned into the BamHI site of a pCMV-81 to produce the mammalianexpression plasmid (provided by Dr. Graeme Bell, Univ. Chicago). Aclonal cell line stably expressing the sst₁ receptor was obtained bytransfection into CHO-K1 cells (ATCC) using the calcium phosphateco-precipitation method (1). The plasmid pRSV-neo (ATCC) was included asa selectable marker. Clonal cell lines were selected in RPMI 1640 mediacontaining 0.5 mg/ml of G418 (Gibco), ring cloned, and expanded intoculture.

The human sst₂ somatostatin receptor gene, isolated as a 1.7 KbBamHI-HindIII genomic DNA fragment and subcloned into the plasmid vectorpGEM3Z (Promega), was kindly provided by Dr. G. Bell (Univ. of Chicago).The mammalian cell expression vector is constructed by inserting the 1.7Kb BamH1-HindII fragment into compatible restriction endonuclease sitesin the plasmid pCMV5. A clonal cell line is obtained by transfectioninto CHO-K1 cells using the calcium phosphate co-precipitation method.The plasmid pRSV-neo is included as a selectable marker.

The human sst₃ was isolated at genomic fragment, and the complete codingsequence was contained within a 2.4 Kb BamHI/HindIII fragment. Themammalian expression plasmid, pCMV-h3 was constructed by inserting the a2.0 Kb NcoI-HindIII fragment into the EcoR1 site of the pCMV vectorafter modification of the ends and addition of EcoR1 linkers. A clonalcell line stably expressing the sst₃ receptor was obtained bytransfection into CHO-K1 cells (ATCC) using the calcium phosphateco-precipitation method. The plasmid pRSV-neo (ATCC) was included as aselectable marker. Clonal cell lines were selected in RPMI 1640 mediacontaining 0.5 mg/ml of G418 (Gibco), ring cloned, and expanded intoculture.

The human sst receptor expression plasmid, pCMV-HX was provided by Dr.Graeme Bell (Univ. Chicago). The vector contains the 1.4 Kb NheI-NheIgenomic fragment encoding the human sst₄, 456 bp of the 5′-untranslatedregion and 200 bp of the 3′-untranslated region, clone into theXbaI/EcoR1 sites of PCMV-HX. A clonal cell line stably expressing thesst₄ receptor was obtained by transfection into CHO-K1 cells (ATCC)using the calcium phosphate co-precipitation method. The plasmidpRSV-neo (ATCC) was included as a selectable marker. Clonal cell lineswere selected in RPMI 1640 media containing 0.5 mg/ml of G418 (Gibco),ring cloned, and expanded into culture.

The human sst₅ gene was obtained by PCR using a λ genomic clone as atemplate, and kindly provided by Dr. Graeme Bell (Univ. Chicago). Theresulting 1.2 Kb PCR fragment contained 21 base pairs of the5′-untranslated region, the full coding region, and 55 bp of the3′-untranslated region. The clone was inserted into EcoR1 site of theplasmid pBSSK(+). The insert was recovered as a 1.2 Kb HindIII-XbaIfragment for subcloning into pCVM5 mammalian expression vector. A clonalcell line stably expressing the SST₅ receptor was obtained bytransfection into CHO-K1 cells (ATCC) using the calcium phosphateco-precipitation method. The plasmid pRSV-neo (ATCC) was included as aselectable marker. Clonal cell lines were selected in RPMI 1640 mediacontaining 0.5 mg/ml of G418 (Gibco), ring cloned, and expanded intoculture.

CHO-K1 cells stably expressing one of the human sst receptor are grownin RPMI 1640 containing 10% fetal calf serum and 0.4 mg/ml geneticin.Cells are collected with 0.5 mM EDTA, and centrifuged at 500 g for about5 min. at about 4° C. The pellet is resuspended in 50 mM Tris, pH 7.4and centrifuged twice at 500 g for about 5 min. at about 4° C. The cellsare lysed by sonication and centrifuged at 39000 g for about 10 min. atabout 4° C. The pellet is resuspended in the same buffer and centrifugedat 50000 g for about 10 min. at about 4° C. and membranes in resultingpellet are stored at −80° C.

Competitive inhibition experiments of (¹²⁵I-Tyr¹¹)SRIF-14 binding arerun in duplicate in polypropylene 96 well plates. Cell membranes (10 μgprotein/well) are incubated with (¹²⁵I-Tyr¹¹)SRIF-14 (0.05 nM) for about60 min. at about 37° C. in 50 mM HEPES (pH 7.4), 0.2% BSA, 5 mM MgCl₂,200 KIU/ml Trasylol, 0.02 mg/ml bacitracin and 0.02 mg/mlphenylmethylsulphonyl fluoride.

Bound from free (¹²⁵I-Tyr¹¹)SRIF-14 is separated by immediate filtrationthrough GF/C glass fiber filter plate (Unifilter, Packard) presoakedwith 0.1% polyethylenimine (P.E.I.), using Filtermate 196 (Packard) cellharvester. Filters are washed with 50 mM HEPES at about 0-4° C. forabout 4 sec. and assayed for radioactivity using Packard Top Count.

Specific binding is obtained by subtracting nonspecific binding(determined in the presence of 0.1 μM SRIF-14) from total binding.Binding data are analyzed by computer-assisted nonlinear regressionanalysis (MDL) and inhibition constant (Ki) values are determined.

The determination of whether a compound of the instant invention is anagonist or an antagonist is determined by the following assay.

Functional assay: Inhibition of CAMP intracellular production:

CHO-K1 Cells expressing human somatostatin (SRIF-14) subtype receptorsare seeded in 24-well tissue culture multidishes in RPMI 1640 media with10% FCS and 0.4 mg/ml geneticin. The medium is changed the day beforethe experiment.

Cells at 10⁵ cells/well are washed 2 times by 0.5 ml and fresh RPMI with0.2% BSA supplemented with 0.5 mM (1) 3-isobutyl-1-methylxanthine (IBMX)and incubated for about 5 min at about 37° C.

-   -   Cyclic AMP production is stimulated by the addition of 1 mM        forskolin (FSK) for about 15-30 minutes at about 37° C.    -   The agonist effect of a compound is measured by the simultaneous        addition of FSK (1 μM), SRIF-14 (10⁻¹² M to 10⁻⁶ M) and a test        compound (10⁻¹⁰ M to 10⁻⁵ M).    -   The antagonist effect of a compound is measured by the        simultaneous addition of FSK (1 μM), SRIF-14 (1 to 10 nM) and a        test compound (10⁻¹⁰ M to 10⁻⁵ M).

The reaction medium is removed and 200 ml 0.1 N HCl is added. CAMP ismeasured using radioimmunoassay method (Kit FlashPlate SMP001A, NewEngland Nuclear).

As is well known to those skilled in the art, the known and potentialuses of somatostatin are varied and multitudinous. Thus, theadministration of a peptide of this invention for purposes ofstimulating the somatostatin receptors can have the same effects or usesas somatostatin itself. For example, inhibiting the secretion of growthhormone, insulin, glucagon and pancreatic exocrine secretion (U.S. Pat.No. 4,853,371); for treating restenosis (U.S. Pat. No. 5,147,856); fortreating hepatoma (U.S. Pat. No. 5,411,943); for treating lung cancer(U.S. Pat. No. 5,073,541); treating melanoma (U.S. Pat. No. 6,087,337issued Jul. 11, 2000; for inhibiting the accelerated growth of a solidtumor (U.S. Pat. No. 5,504,069); for decreasing body weight (WO 98/51331published Nov. 19, 1998); for treating insulin resistance and Syndrome X(WO 98/51332 published Nov. 19, 1998); for prolonging the survival ofpancreatic cells (U.S. Pat. No. 5,688,418); for treating fibrosis (WO98/08529 published Mar. 5, 1998); for treating hyperlipidemia (WO98/51330 published Nov. 19, 1998); for treating hyperamylinemia (U.S.Pat. No. 5,763,200 issued Jun. 9, 1998); for treating hyperprolactinemiaand prolactinomas (U.S. Pat. No. 5,972,893 issued Oct. 26, 1999);Cushings Syndrome (see Clark, R. V. et al, Clin. Res. 38, p. 943A,1990); gonadotropinoma (see Ambrosi B., et al., Acta Endocr. (Copenh.)122, 569-576, 1990); hyperparathyroidism (see Miller, D., et al., Canad.Med. Ass. J., Vol. 145, pp. 227-228, 1991); Paget's disease (see,Palmieri, G. M. A., et al., J. of Bone and Mineral Research, 7, (Suppl.1), p. S240 (Abs. 591), 1992); VIPoma (see Koberstein, B., et al., Z.Gastroenterology, 28, 295-301, 1990 and Christensen, C., Acta Chir.Scand. 155, 541-543, 1989); nesidioblastosis and hyperinsulinism (seeLaron, Z., Israel J. Med. Sci., 26, No. 1, 1-2, 1990, Wilson, D. C.,Irish J. Med. Sci., 158, No. 1, 31-32, 1989 and Micic, D., et al.,Digestion, 16, Suppl. 1.70. Abs. 193, 1990); gastrinoma (see Bauer, F.E., et al., Europ. J. Pharmacol., 183, 55 1990); Zollinger-EllisonSyndrome (see Mozell, E., et al., Surg. Gynec. Obstet., 170, 476-484,1990); hypersecretory diarrhea related to AIDS and other conditions (dueto AIDS, see Cello, J. P., et al., Gastroenterology, 98, No. 5, Part 2,Suppl., A163 1990; due to elevated gastrin-releasing peptide, seeAlhindawi, R., et al., Can. J. Surg., 33, 139-142, 1990; secondary tointestinal graft vs. host disease, see Bianco J. A., et al.,Transplantation, 49, 1194-1195, 1990; diarrhea associated withchemotherapy, see Petrelli, N., et al., Proc. Amer. Soc. Clin. Oncol.,Vol. 10, P 138, Abstr. No. 417 1991); irritable bowel syndrome (seeO'Donnell, L. J. D., et al., Aliment. Pharmacol. Therap., Vol. 4.,177-181, 1990); pancreatitis (see Tulassay, Z., et al.,Gastroenterology, 98, No. 5, Part 2, Suppl., A238, 1990); Crohn'sDisease (see Fedorak, R. N., et al., Can. J. Gastroenterology, 3, No.2,53-57, 1989); systemic sclerosis (see Soudah, H., et al.,Gastroenterology, 98, No. 5, Part 2, Suppl., A129, 1990); thyroid cancer(see Modigliani, E., et al., Ann., Endocr. (Paris), 50, 483-488, 1989);psoriasis (see Camisa, C., et al., Cleveland Clinic J. Med., 57, No. 1,71-76, 1990); hypotension (see Hoeldtke, R. D., et al., Arch. Phys. Med.Rehabil., 69, 895-898, 1988 and Kooner, J. S., et al., Brit. J. Clin.Pharmacol., 28, 735P-736P, 1989); panic attacks (see Abelson, J. L., etal., Clin. Psychopharmacol., 10, 128-132, 1990); sclerodoma (see Soudah,H., et al., Clin. Res., Vol.39, p. 303A, 1991); small bowel obstruction(see Nott, D. M., et al., Brit. J. Surg., Vol. 77, p. A691, 1990);gastroesophageal reflux (see Branch, M. S., et al., Gastroenterology,Vol. 100, No. 5, Part 2 Suppl., p. A425, 1991); duodenogastric reflux(see Hasler, W., et al., Gastroenterology, Vol. 100, No.5, Part 2,Suppl., p. A448, 1991); Graves' Disease (see Chang, T. C., et al., Brit.Med. J., 304, p. 158, 1992); polycystic ovary disease (see Prelevic, G.M., et al., Metabolism Clinical and Experimental, 41, Suppl. 2, pp76-79, 1992); upper gastrointestinal bleeding (see Jenkins, S. A., etal., Gut., 33, pp. 404-407, 1992 and Arrigoni, A., et al., AmericanJournal of Gastroenterology, 87, p. 1311, (abs. 275), 1992); pancreaticpseudocysts and ascites (see Hartley, J. E., et al., J. Roy. Soc. Med.,85, pp. 107-108, 1992); leukemia (see Santini, et al., 78, (Suppl. 1),p. 429A (Abs. 1708), 1991); meningioma (see Koper, J. W., et al., J.Clin. Endocr. Metab., 74, pp. 543-547, 1992); and cancer cachexia (seeBartlett, D. L., et al., Surg. Forum., 42, pp. 14-16, 1991).

Accordingly, the present invention includes within its scopepharmaceutical compositions comprising, as an active ingredient, atleast one of the peptides of formula (I) in association with apharmaceutically acceptable carrier.

In general an effective dosage for the activities of this invention, forexample the treatment of acromegaly, is in the range of 0.01 to 200mg/kg/day, preferably 0.5 to 100mg/kg/day.

A peptide of this invention can be administered by oral, parenteral(e.g., intramuscular, intraperitoneal, intravenous or subcutaneousinjection, or implant), nasal, vaginal, rectal, sublingual or topicalroutes of administration and can be formulated with pharmaceuticallyacceptable carriers to provide dosage forms appropriate for each routeof administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than suchinert diluents, e.g., lubricating agents such as magnesium stearate. Inthe case of capsules, tablets and pills, the dosage forms may alsocomprise buffering agents. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as coca butter or a suppository wax.

Compositions for nasal or sublingual administration are also preparedwith standard excipients well known in the art.

Further, a compound of this invention can be administered in a sustainedrelease composition such as those described in the following patents andpatent applications. U.S. Pat. No. 5,672,659 teaches sustained releasecompositions comprising a bioactive agent and a polyester. U.S. Pat. No.5,595,760 teaches sustained release compositions comprising a bioactiveagent in a gelable form. U.S. Pat. No. 5,821,221, teaches polymericsustained release compositions comprising a bioactive agent andchitosan. U.S. Pat. No. 5,916,883, issued Jun. 26, 1999, teachessustained release compositions comprising a bioactive agent andcyclodextrin. International patent publication WO 99/38536 publishedAug. 5, 1999, teaches absorbable sustained release compositions of abioactive agent. U.S. Pat. No. 6,270,700, teaches a process for makingmicroparticles comprising a therapeutic agent such as a peptide in anoil-in-water process. International patent publication WO 00/09166,published Feb. 24, 2000, teaches complexes comprising a therapeuticagent such as a peptide and a phosphorylated polymer. Internationalpatent publication WO 00/25826 published May 11, 2000, teaches complexescomprising a therapeutic agent such as a peptide and a polymer bearing anon-polymerizable lactone. The teachings of the foregoing patents andapplications are incorporated herein by reference.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient be such that a suitable dosage form is obtained. The selecteddosage depends upon the desired therapeutic effect, on the route ofadministration, and on the duration of the treatment. Generally, dosagelevels of between 0.0001 to 100 mg/kg of body weight daily areadministered to humans and other animals, e.g., mammals, to obtaineffective release of growth hormone.

A preferred dosage range is 0.01 to 5.0 mg/kg of body weight daily whichcan be administered as a single dose or divided into multiple doses.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Also, all publications, patentapplications, patents, and other references mentioned herein areincorporated by reference.

1. A method of eliciting a somatostatin agonist response in a human orother animal in need thereof, which comprises administering an effectiveamount of a peptide of formula (I)X-A¹-cyclo(D-Cys-A³-A⁴-Lys-A⁶-A⁷)-A⁸-Y,  (I) or a pharmaceuticallyacceptable salt thereof, wherein

A¹ and A³ are each independently the D- or L-isomer of an amino acidselected from the group consisting of Phe, Tyr, Tyr(I), Trp, 3-Pal,4-Pal, Cpa and Nal; A⁴ is L-Trp, D-Trp, L-β-methyl-Trp orD-β-methyl-Trp; A⁶ is —NH—(CHR¹)_(n)—CO—, where n is 2, 3, or 4; A⁷ isL- or D-Cys; A⁸ is the D- or L-isomer of an amino acid selected from thegroup consisting of Phe, Tyr, Tyr(I), Trp, Nal, Cpa, Val, Leu, Ile, Serand Thr; Y is NR²R³ where R² and R³ are each independently H or(C₁-C₅)alkyl; R¹ is selected from the group consisting H, (C₁-C₄)alkyland —CH₂-aryl; wherein said aryl is an optionally substituted moietyselected from the group consisting of phenyl, 1-naphthyl, and2-naphthyl, wherein said optionally substituted moiety is optionallysubstituted with one or more substituents each independently selectedfrom the group consisting of (C₁₋₆)alkyl, (C₂₋₆)alkenyl, (C₂₋₆)alkynyl,aryl, aryl(C₁₋₆)alkyl, (C₁₋₆)alkoxy, —N(R⁴R⁵), —COOH, —CON(R⁴R⁵), halo,—OH, —CN, and —NO₂; R⁴ and R⁵ each is, independently for eachoccurrence, H or (C₁₋₃)alkyl; where the Cys of A² is bonded to the Cysof A⁷ by a di-sulfide bond formed from the thiol groups of each Cys, tothe human or other animal.
 2. A method of inhibiting the secretion ofgrowth hormone, insulin, glucagon or pancreatic exocrine secretion in ahuman or other animal in need thereof, which comprises administering apeptide of formula (I)X-A¹-cyclo(D-Cys-A³-A⁴-Lys-A⁶-A⁷)-A⁸-Y,  (I) or a pharmaceuticallyacceptable salt thereof, wherein

A¹ and A³ are each independently the D- or L-isomer of an amino acidselected from the group consisting of Phe, Tyr, Tyr(I), Trp, 3-Pal,4-Pal, Cpa and Nal; A⁴ is L-Trp, D-Trp, L-β-methyl-Trp orD-β-methyl-Trp; A⁶ is —NH—(CHR¹)_(n)—CO—, where n is 2, 3, or 4; A⁷ isL- or D-Cys; A⁸ is the D- or L-isomer of an amino acid selected from thegroup consisting of Phe, Tyr, Tyr(I), Trp, Nal, Cpa, Val, Leu, Ile, Serand Thr; Y is NR²R³ where R² and R³ are each independently H or(C₁-C₅)alkyl; R¹ is selected from the group consisting H, (C₁-C₄)alkyland —CH₂-aryl; wherein said aryl is an optionally substituted moietyselected from the group consisting of phenyl, 1-naphthyl, and2-naphthyl, wherein said optionally substituted moiety is optionallysubstituted with one or more substituents each independently selectedfrom the group consisting of (C₁₋₆)alkyl, (C₂₋₆)alkenyl, (C₂₋₆)alkynyl,aryl, aryl(C₁₋₆)alkyl, (C₁₋₆)alkoxy, —N(R⁴R⁵), —COOH, —CON(R⁴R⁵), halo,—OH, —CN, and —NO₂; R⁴ and R⁵ each is, independently for eachoccurrence, H or (C₁₋₃)alkyl; where the Cys of A² is bonded to the Cysof A⁷ by a di-sulfide bond formed from the thiol groups of each Cys, tosaid human or other animal.
 3. The method according to claim 1 or 2,wherein said peptide of formula (I) is a peptide wherein A¹ is L-Phe,D-Phe, L-Cpa or D-Cpa; A³ is L-Tyr, L-Trp or L-3-Pal; A⁴ is D-Trp; A⁶ isβ-Ala or Gaba; A⁷ is L-Cys; A⁸ is L-Thr, L-Trp, L-Leu or L-Nal; and R²and R³ are each H; or a pharmaceutically acceptable salt thereof.
 4. Themethod according to claim 1 or 2, wherein said peptide is of the formulaCpa-cyclo(D-Cys-3-Pal-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;Cpa-cyclo(D-Cys-3-Pal-D-Trp-Lys-β-Ala-Cys)-Nal-NH₂;Phe-cyclo(D-Cys-3-Pal-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;Phe-cyclo(D-Cys-Trp-D-Trp-Lys-Gaba-Cys)-Nal-NH₂;Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Trp-NH₂;D-Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)Nal-NH₂;D-Phe-cyclo(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Leu-NH₂; orPhe-cyclo-(D-Cys-Tyr-D-Trp-Lys-Gaba-Cys)-Thr-NH₂; or a pharmaceuticallyacceptable salt thereof.
 5. The method according to claim 1 or 2,wherein said peptide is of the formulaCpa-cyclo(D-Cys-3-Pal-D-Trp-Lys-Gaba-Cys)-Nal-NH₂; orCpa-cyclo(D-Cys-3-Pal-D-Trp-Lys-β-Ala-Cys)-Nal-NH₂; or apharmaceutically acceptable salt thereof.
 6. The method according toclaim 1 or 2, wherein said peptide or pharmaceutically acceptable saltthereof is in the form of a pharmaceutical composition which comprisesan effective amount of a peptide of formula (I) or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.